Methods for the identification of inhibitors of threonine synthase as antibiotics

ABSTRACT

The present inventors have discovered that Threonine synthase is essential for fungal pathogenicity. Specifically, the inhibition of Threonine synthase gene expression in fungi results in no signs of successful infection or lesions. Thus, Threonine synthase can be used as a target for the identification of antibiotics, preferably antifungals. Accordingly, the present invention provides methods for the identification of compounds that inhibit Threonine synthase expression or activity. The methods of the invention are useful for the identification of antibiotics, preferably antifungals.

FIELD OF THE INVENTION

The invention relates generally to methods for the identification ofantibiotics, preferably antifungals that affect the biosynthesis ofL-threonine.

BACKGROUND OF THE INVENTION

Filamentous fungi are the causal agents responsible for many seriouspathogenic infections of plants and animals. Since fungi are eukaryotes,and thus more similar to their host organisms than, for examplebacteria, the treatment of infections by fungi poses special risks andchallenges not encountered with other types of infections. One suchfungus is Magnaporthe grisea, the fungus that causes rice blast disease.It is an organism that poses a significant threat to food suppliesworldwide. Other examples of plant pathogens of economic importanceinclude the pathogens in the genera Agaricus, Alternaria, Anisogramma,Anthracoidea, Antrodia, Apiognomonia, Apiosporina, Armillaria,Ascochyta, Aspergillus, Bipolaris, Bjerkandera, Botryosphaeria,Botrytis, Ceratobasidium, Ceratocystis, Cercospora, Cercosporidium,Cerotelium, Cerrena, Chondrostereum, Chryphonectria, Chrysomyxa,Cladosporium, Claviceps, Cochliobolus, Coleosporium, Colletotrichium,Colletotrichum, Corticium, Corynespora, Cronartium, Cryphonectria,Cryptosphaeria, Cyathus, Cymadothea, Cytospora, Daedaleopsis, Diaporthe,Didymella, Diplocarpon, Diplodia, Discohainesia, Discula, Dothistroma,Drechslera, Echinodontium, Elsinoe, Endocronartium, Endothia, Entyloma,Epichloe, Erysiphe, Exobasidium, Exserohilum, Fomes, Fomitopsis,Fusarium, Gaeumannomyces, Ganoderma, Gibberella, Gloeocercospora,Gloeophyllum, Gloeoporus, Glomerella, Gnomoniella, Guignardia,Gymnosporangium, Helminthosporium, Herpotrichia, Heterobasidion,Hirschioporus, Hypodermella, Inonotus, Irpex, Kabatiella, Kabatina,Laetiporus, Laetisaria, Lasiodiplodia, Laxitextum, Leptographium,Leptosphaeria, Leptosphaerulina, Leucytospora, Linospora, Lophodermella,Lophodermium, Macrophomina, Magnaporthe, Marssonina, Melampsora,Melampsorella, Meria, Microdochium, Microsphaera, Monilinia,Monochaetia, Morchella, Mycosphaerella, Myrothecium, Nectria,Nigrdspora, Ophiosphaerella, Ophiostoma, Penicillium, Perenniporia,Peridermium, Pestalotia, Phaeocryptopus, Phaeolus, Phakopsora,Phellinus, Phialophora, Phoma, Phomopsis, Phragmidium, Phyllachora,Phyllactinia, Phyllosticta, Phymatotrichopsis, Pleospora, Podosphaera,Pseudopeziza, Pseudoseptoria, Puccinia, Pucciniastrum, Pyricularia,Rhabdocline, Rhizoctonia, Rhizopus, Rhizosphaera, Rhynchosporium,Rhytisma, Schizophyllum, Schizopora, Scirrhia, Sclerotinia, Sclerotium,Scytinostroma, Septoria, Setosphaera, Sirococcus, Spaerotheca,Sphaeropsis, Sphaerotheca, Sporisorium, Stagonospora, Stemphylium,Stenocarpella, Stereum, Taphrina, Thielaviopsis, Tilletia, Trametes,Tranzschelia, Trichoderma, Tubakia, Typhula, Uncinula, Urocystis,Uromyces, Ustilago, Valsa, Venturia, Verticillium, Xylaria, and others.Related organisms in the classification, oomycetes, that include thegenera Albugo, Aphanomyces, Bremia, Peronospora, Phytophthora,Plasmodiophora, Plasmopara, Pseudoperonospora, Pythium, Sclerophthora,and others are also significant plant pathogens and are sometimesclassified along with the true fungi. Human diseases that are caused byfilamentous fungi include life-threatening lung and disseminateddiseases, often a result of infections by Aspergillus fumigatus. Otherfungal diseases in animals are caused by fungi in the genera, Fusarium,Blastomyces, Microsporum, Trichophyton, Epidermophyton, Candida,Histoplamsa, Pneumocystis, Cryptococcus, other Aspergilli, and others.The control of fungal diseases in plants and animals is usually mediatedby chemicals that inhibit the growth, proliferation, and/orpathogenicity of the fungal organisms. To date, there are less thantwenty known modes-of-action for plant protection fungicides and humanantifungal compounds.

A pathogenic organism has been defined as an organism that causes, or iscapable of causing disease. Pathogenic organisms propagate on or intissues and may obtain nutrients and other essential materials fromtheir hosts. A substantial amount of work concerning filamentous fungalpathogens has been performed with the human pathogen, Aspergillusfumigatus. Shibuya et al. (Shibuya, K., M. Takaoka, et al. (1999) MicrobPathog 27: 123-31 (PMID: 10455003)) have shown that the deletion ofeither of two suspected pathogenicity related genes encoding an alkalineprotease or a hydrophobin (rodlet) respectively, did not reducemortality of mice infected with these mutant strains. Smith et al.(Smith, J. M., C. M. Tang, et al. (1994) Infect Immun 62: 5247-54 (PMID:7960101)) showed similar results with alkaline protease and theribotoxin restrictocin; Aspergillus fumigatus strains mutated for eitherof these genes were fully pathogenic to mice. Reichard et al. (Reichard,U., M. Monod, et al. (1997) J Med Vet Mycol 35: 189-96 (PMID: 9229335))showed that deletion of the suspected pathogenicity gene encodingaspergillopepsin (PEP) in Aspergillus fumigatus had no effect onmortality in a guinea pig model system, and Aufauvre-Brown et al(Aufauvre-Brown, A., E. Mellado, et al. (1997) Fungal Genet Biol 21:141-52 (PMID: 9073488)) showed no effects of a chitin synthase mutationon pathogenicity. However, not all experiments produced negativeresults. Ergosterol is an important membrane component found in fungalorganisms. Pathogenic fungi that lack key enzymes in this biochemicalpathway might be expected to be non-pathogenic since neither the plantnor animal hosts contain this particular sterol. Many antifungalcompounds that affect this biochemical pathway have been described(Onishi, J. C. and A. A. Patchett (1990a, b, c, d, and e) U.S. Pat. Nos.4,920,109; 4,920,111; 4,920,112; 4,920,113; and 4,921,844, Merck & Co.Inc. (Rahway N.J.)) and (Hewitt, H. G. (1998) Fungicides in CropProtection Cambridge, University Press). D'Enfert et al. (D'Enfert, C.,M. Diaquin, et al. (1996) Infect Immun 64: 4401-5 (PMID: 8926121))showed that an Aspergillus fumigatus strain mutated in an orotidine5′-phosphate decarboxylase gene was entirely non-pathogenic in mice, andBrown et al. (Brown, J. S., A. Aufauvre-Brown, et al. (2000) MolMicrobiol 36: 1371-80 (PMID: 10931287)) observed a non-pathogenic resultwhen genes involved in the synthesis of para-aminobenzoic acid weremutated. Some specific target genes have been described as havingutility for the screening of inhibitors of plant pathogenic fungi. Bacotet al. (Bacot, K. O., D. B. Jordan, et al. (2000) U.S. Pat. No.6,074,830, E. I. du Pont de Nemours & Company (Wilmington Del.))describe the use of 3,4-dihydroxy-2-butanone 4-phosphate synthase, andDavis et al. (Davis, G. E., G. D. Gustafson, et al. (1999) U.S. Pat. No.5,976,848, Dow AgroSciences LLC (Indianapolis Ind.)) describe the use ofdihydroorotate dehydrogenase for potential screening purposes.

There are also a number of papers that report less clear results,showing neither full pathogenicity nor non-pathogenicity of mutants.Hensel et al. (Hensel, M., H. N. Arst, Jr., et al. (1998) Mol Gen Genet258: 553-7 (PMID: 9669338)) showed only moderate effects of the deletionof the areA transcriptional activator on the pathogenicity ofAspergillus fumigatus.

Therefore, it is not currently possible to determine which specificgrowth materials may be readily obtained by a pathogen from its host,and which materials may not. We have found that Magnaporthe grisea thatcannot synthesize their own L-threonine are non-pathogenic on their hostorganism. To date there do not appear to be any publicationsdemonstrating an anti-pathogenic effect of the knock-out,over-expression, antisense expression, or inhibition of the genes orgene products involved in L-threonine biosynthesis in filamentous fungi.Thus, it has not been shown that the de novo biosynthesis of L-threonineis essential for fungal pathogenicity. Thus, it would be desirable todetermine the utility of the enzymes involved in L-threoninebiosynthesis for evaluating antibiotic compounds, especially fungicides.If a fungal biochemical pathway or specific gene product in that pathwayis shown to be required for fungal pathogenicity, various formats of invitro and in vivo screening assays may be put in place to discoverclasses of chemical compounds that react with the validated target gene,gene product, or biochemical pathway, and are thus candidates forantifungal, biocide, and biostatic materials.

SUMMARY OF THE INVENTION

Surprisingly, the present inventors have discovered that in vivodisruption of the gene encoding Threonine synthase in Magnaporthe griseaprevents or inhibits the pathogenicity of the fungus. Thus, the presentinventors have discovered that Threonine synthase is essential fornormal rice blast pathogenicity, and can be used as a target for theidentification of antibiotics, preferably fungicides. Accordingly, thepresent invention provides methods for the identification of compoundsthat inhibit Threonine synthase expression or activity. The methods ofthe invention are useful for the identification of antibiotics,preferably fungicides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the reaction performed by Threonine synthase (THR4)reaction. The Substrates/Products are O-phospho-L-homoserine and waterand the Products/Substrates are L-threonine and orthophosphate. Thefunction of the Threonine synthase enzyme is the interconversion ofO-phospho-L-homoserine and water to L-threonine and orthophosphate. Thisreaction is part of the L-threonine biosynthesis pathway.

FIG. 2 shows a digital image showing the effect of THR4 gene disruptionon Magnaporthe grisea pathogenicity using whole plant infection assays.Rice variety CO39 was inoculated with wild-type and the transposoninsertion strains, KO1-3 and KO1-22. Leaf segments were imaged at fivedays post-inoculation.

FIG. 3. Verification of Gene Function by Analysis of NutritionalRequirements. Wild-type and transposon insertion strains, KO1-3 andKO1-22, were grown in (A) minimal media and (B) minimal media with theaddition of L-threonine, respectively. The x-axis shows time in days andthe y-axis shows turbidity measured at 490 nanometers and 750nanometers. The symbols represent wildtype (--♦--), transposon strainKO1-3 (--▪--), and transposon strain KO1-22 (--▴--).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, the following terms are intended to have thefollowing meanings in interpreting the present invention.

The term “active against” in the context of compounds, agents, orcompositions having antibiotic activity indicates that the compoundexerts an effect on a particular target or targets which is deleteriousto the in vitro and/or in vivo growth of an organism having that targetor targets. In particular, a compound active against a gene exerts anaction on a target which affects an expression product of that gene.This does not necessarily mean that the compound acts directly on theexpression product of the gene, but instead indicates that the compoundaffects the expression product in a deleterious manner. Thus, the directtarget of the compound may be, for example, at an upstream componentwhich reduces transcription from the gene, resulting in a lower level ofexpression. Likewise, the compound may affect the level of translationof a polypeptide expression product, or may act on a downstreamcomponent of a biochemical pathway in which the expression product ofthe gene has a major biological role. Consequently, such a compound canbe said to be active against the gene, against the gene product, oragainst the related component either upstream or downstream of that geneor expression product. While the term “active against” encompasses abroad range of potential activities, it also implies some degree ofspecificity of target. Therefore, for example, a general protease is not“active against” a particular gene which produces a polypeptide product.In contrast, a compound which inhibits a particular enzyme is activeagainst that enzyme and against the gene which codes for that enzyme.

As used herein, the term “allele” refers to any of the alternative formsof a gene that may occur at a given locus.

The term “antibiotic” refers to any substance or compound that whencontacted with a living cell, organism, virus, or other entity capableof replication, results in a reduction of growth, viability, orpathogenicity of that entity.

The term “binding” refers to a non-covalent or a covalent interaction,preferably non-covalent, that holds two molecules together. For example,two such molecules could be an enzyme and an inhibitor of that enzyme.Non-covalent interactions include hydrogen bonding, ionic interactionsamong charged groups, van der Waals interactions and hydrophobicinteractions among nonpolar groups. One or more of these interactionscan mediate the binding of two molecules to each other.

The term “biochemical pathway” or “pathway” refers to a connected seriesof biochemical reactions normally occurring in a cell, or more broadly acellular event such as cellular division or DNA replication. Typically,the steps in such a biochemical pathway act in a coordinated fashion toproduce a specific product or products or to produce some otherparticular biochemical action. Such a biochemical pathway requires theexpression product of a gene if the absence.of that expression producteither directly or indirectly prevents the completion of one or moresteps in that pathway, thereby preventing or significantly reducing theproduction of one or more normal products or effects of that pathway.Thus, an agent specifically inhibits such a biochemical pathwayrequiring the expression product of a particular gene if the presence ofthe agent stops or substantially reduces the completion of the series ofsteps in that pathway. Such an agent, may, but does not necessarily, actdirectly on the expression product of that particular gene.

As used herein, the term “cDNA” means complementary deoxyribonucleicacid.

As used herein, the term “CoA” means coenzyme A.

As used herein, the term “conditional lethal” refers to a mutationpermitting growth and/or survival only under special growth orenvironmental conditions.

As used herein, the term “cosmid” refers to a hybrid vector, used ingene cloning, that includes a cos site (from the lambda bacteriophage).It also contains drug resistance marker genes and other plasmid genes.Cosmids are especially suitable for cloning large genes or multigenefragments.

As used herein, the term “dominant allele” refers to a dominant mutantallele in which a discernable mutant phenotype can be detected when thismutation is present in an organism that also contains a wild type(non-mutant), recessive allele, or other dominant allele.

As used herein, the term “DNA” means deoxyribonucleic acid.

As used herein, the term “ELISA” means enzyme-linked immunosorbentassay.

“Fungi” (singular: fungus) refers to whole fungi, fungal organs andtissues (e.g., asci, hyphae, pseudohyphae, rhizoid, scierotia,sterigmata, spores, sporodochia, sporangia, synnemata, conidia,ascostroma, cleistothecia, mycelia, perithecia, basidia and the like),spores, fungal cells and the progeny thereof. Fungi are a group oforganisms (about 50,000 known species), including, but not limited to,mushrooms, mildews, moulds, yeasts, etc., comprising the kingdom Fungi.They can either exist as single cells or make up a multicellular bodycalled a mycelium, which consists of filaments known as hyphae. Mostfungal cells are multinucleate and have cell walls, composed chiefly ofchitin. Fungi exist primarily in damp situations on land and, because ofthe absence of chlorophyll and thus the inability to manufacture theirown food by photosynthesis, are either parasites on other organisms orsaprotrophs feeding on dead organic matter. The principal criteria usedin classification are the nature of the spores produced and the presenceor absence of cross walls within the hyphae. Fungi are distributedworldwide in terrestrial, freshwater, and marine habitats. Some live inthe soil. Many pathogenic fungi cause disease in animals and man or inplants, while some saprotrophs are destructive to timber, textiles, andother materials. Some fungi form associations with other organisms, mostnotably with algae to form lichens.

As used herein, the term “fungicide”, “antifungal”, or “antimycotic”refers to an antibiotic substance or compound that kills or suppressesthe growth, viability, or pathogenicity of at least one fungus, fungalcell, fungal tissue or spore.

In the context of this disclosure, “gene” should be understood to referto a unit of heredity. Each gene is composed of a linear chain ofdeoxyribonucleotides which can be referred to by the sequence ofnucleotides forming the chain. Thus, “sequence” is used to indicate boththe ordered listing of the nucleotides which form the chain, and thechain, itself, which has that sequence of nucleotides. (“Sequence” isused in the similar way in referring to RNA chains, linear chains madeof ribonucleotides). The gene may include regulatory and controlsequences, sequences which can be transcribed into an RNA molecule, andmay contain sequences with unknown function. The majority of the RNAtranscription products are messenger RNAs (mRNAs), which includesequences which are translated into polypeptides and may includesequences which are not translated. It should be recognized that smalldifferences in nucleotide sequence for the same gene can exist betweendifferent fungal strains, or even within a particular fungal strain,without altering the identity of the gene.

As used in this disclosure, the terms “growth” or “cell growth” of anorganism refers to an increase in mass, density, or number of cells ofsaid organism. Some common methods for the measurement of growth includethe determination of the optical density of a cell suspension, thecounting of the number of cells in a fixed volume, the counting of thenumber of cells by measurement of cell division, the measurement ofcellular mass or cellular volume, and the like.

As used in this disclosure, the term “growth conditional phenotype”indicates that a fungal strain having such a phenotype exhibits asignificantly greater difference in growth rates in response to a changein one or more of the culture parameters than an otherwise similarstrain not having a growth conditional phenotype. Typically, a growthconditional phenotype is described with respect to a single growthculture parameter, such as temperature. Thus, a temperature (orheat-sensitive) mutant (i.e., a fungal strain having a heat-sensitivephenotype) exhibits significantly different growth, and preferably nogrowth, under non-permissive temperature conditions as compared togrowth under permissive conditions. In addition, such mutants preferablyalso show intermediate growth rates at intermediate, or semi-permissive,temperatures. Similar responses also result from the appropriate growthchanges for other types of growth conditional phenotypes.

As used herein, the term “H₂O” means water.

As used herein, the term “heterologous THR4 gene” means a gene, notderived from Magnaporthe grisea, and having: at least 50% sequenceidentity, preferably 60%, 70%, 80%, 90%, 95%, 99% sequence identity andeach integer unit of sequence identity from 50-100% in ascending orderto SEQ ID NO: 1 or SEQ ID NO: 2; or at least 10% of the activity of aMagnaporthe grisea Threonine synthase, preferably 25%, 50%, 75%, 90%,95%, 99% and each integer unit of activity from 10-100% in ascendingorder.

As used herein, the term “His-Tag” refers to an encoded polypeptideconsisting of multiple consecutive histidine amino acids.

As used herein, the term “HPLC” means high pressure liquidchromatography.

As used herein, the terms “hph”, “hygromycin B phosphotransferase”, and“hygromycin resistance gene” refer to the E. coli hygromycinphosphotransferase gene or gene product.

As used herein, the term “hygromycin B” refers to an aminoglycosidicantibiotic, used for selection and maintenance of eukaryotic cellscontaining the E. coli hygromycin resistance gene.

“Hypersensitive” refers to a phenotype in which cells are more sensitiveto antibiotic compounds than are wild-type cells of similar or identicalgenetic background.

“Hyposensitive” refers to a phenotype in which cells are less sensitiveto antibiotic compounds than are wild-type cells of similar or identicalgenetic background.

As used herein, the term “imperfect state” refers to a classification ofa fungal organism having no demonstrable sexual life stage.

The term “inhibitor”, as used herein, refers to a chemical substancethat inactivates the enzymatic activity of Threonine synthase orsubstantially reduces the level of enzymatic activity, wherein“substantially” means a reduction at least as great as the standarddeviation for a measurement, preferably a reduction by 50%, morepreferably a reduction of at least one magnitude, i.e. to 10%. Theinhibitor may function by interacting directly with the enzyme, acofactor of the enzyme, the substrate of the enzyme, or any combinationthereof.

A polynucleotide may be “introduced” into a fungal cell by any meansknown to those of skill in the art, including transfection,transformation or transduction, transposable element, electroporation,particle bombardment, infection and the like. The introducedpolynucleotide may be maintained in the cell stably if it isincorporated into a non-chromosomal autonomous replicon or integratedinto the fungal chromosome. Alternatively, the introduced polynucleotidemay be present on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active.

As used herein, the term “knockout” or “gene disruption” refers to thecreation of organisms carrying a null mutation (a mutation in whichthere is no active gene product), a partial null mutation or mutations,or an alteration or alterations in gene regulation by interrupting a DNAsequence through insertion of a foreign piece of DNA. Usually theforeign DNA encodes a selectable marker.

As used herein, the term “LB agar” means Luria's Broth agar.

The term “method of screening” means that the method is suitable, and istypically used, for testing for a particular property or effect in alarge number of compounds. Typically, more than one compound is testedsimultaneously (as in a 96-well microtiter plate), and preferablysignificant portions of the procedure can be automated. “Method ofscreening” also refers to the determination of a set of differentproperties or effects of one compound simultaneously.

As used herein, the term “mRNA” means messenger ribonucleic acid.

As used herein, the term “mutant form” of a gene refers to a gene whichhas been altered, either naturally or artificially, changing the basesequence of the gene. The change in the base sequence may be of severaldifferent types, including changes of one or more bases for differentbases, deletions, and/or insertions, such as by a transposon. Bycontrast, a normal form of a gene (wild type) is a form commonly foundin natural populations of an organism. Commonly a single form of a genewill predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

As used herein, the term “Ni” refers to nickel.

As used herein, the term “Ni-NTA” refers to nickel sepharose.

As used herein, a “normal” form of a gene (wild type) is a form commonlyfound in natural populations of an organism. Commonly a single form of agene will predominate in natural populations. In general, such a gene issuitable as a normal form of a gene, however, other forms which providesimilar functional characteristics may also be used as a normal gene. Inparticular, a normal form of a gene does not confer a growth conditionalphenotype on the strain having that gene, while a mutant form of a genesuitable for use in these methods does provide such a growth conditionalphenotype.

As used herein, the term “one form” of a gene is synonymous with theterm “gene”, and a “different form” of a gene refers to a gene that hasgreater than 49% sequence identity and less than 100% sequence identitywith said first form.

As used herein, the term “pathogenicity” refers to a capability ofcausing disease. The term is applied to parasitic microorganisms inrelation to their hosts.

As used herein, the term “PCR” means polymerase chain reaction.

The “percent (%) sequence identity” between two polynucleotide or twopolypeptide sequences is determined according to the either the BLASTprogram (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish,et al. (1990) J Mol Biol 215: 403-10 (PMID: 2231712)) at the NationalCenter for Biotechnology or using Smith Waterman Alignment (Smith, T. F.and M. S. Waterman (1981) J Mol Biol 147: 195-7 (PMID: 7265238)) asincorporated into GeneMatcher Plus™. It is understood that for thepurposes of determining sequence identity when comparing a DNA sequenceto an RNA sequence, a thymine nucleotide is equivalent to a uracilnucleotide.

By “polypeptide” is meant a chain of at least two amino acids joined bypeptide bonds. The chain may be linear, branched, circular orcombinations thereof. Preferably, polypeptides are from about 10 toabout 1000 amino acids in length, more preferably 10-50 amino acids inlength. The polypeptides may contain amino acid analogs and othermodifications, including, but not limited to glycosylated orphosphorylated residues.

As used herein, the term “proliferation” is synonymous to the term“growth”.

As used herein, the term “reverse transcriptase-PCR” means reversetranscription-polymerase chain reaction.

As used herein, the term “RNA” means ribonucleic acid.

As used herein, “semi-permissive conditions” are conditions in which therelevant culture parameter for a particular growth conditional phenotypeis intermediate between permissive conditions and non-permissiveconditions. Consequently, in semi-permissive conditions an organismhaving a growth conditional phenotype will exhibit growth ratesintermediate between those shown in permissive conditions andnon-permissive conditions. In general, such intermediate growth rate maybe due to a mutant cellular component which is partially functionalunder semi-permissive conditions, essentially fully functional underpermissive conditions, and is non-functional or has very low functionunder non-permissive conditions, where the level of function of thatcomponent is related to the growth rate of the organism. An intermediategrowth rate may also be a result of a nutrient substance or substancesthat are present in amounts not sufficient for optimal growth rates tobe achieved.

“Sensitivity phenotype” refers to a phenotype that exhibits eitherhypersensitivity or hyposensitivity.

The term “specific binding” refers to an interaction between Threoninesynthase and a molecule or compound, wherein the interaction isdependent upon the primary amino acid sequence and/or the conformationof Threonine synthase.

As used herein, the term “THR4” means a gene encoding Threonine synthaseactivity, referring to an enzyme that catalyses the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphate,and may also be used to refer to the gene product.

As used herein, the terms “Threonine synthase” (EC 4.2.99.2) and“Threonine synthase polypeptide” are synonymous with “the THR4 geneproduct” and refer to an enzyme that catalyses the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphate.

As used herein, the term “TLC” means thin layer chromatography.

“Transform”, as used herein, refers to the introduction of apolynucleotide (single or double stranded DNA, RNA, or a combinationthereof) into a living cell by any means. Transformation may beaccomplished by a variety of methods, including, but not limited to,electroporation, polyethylene glycol mediated uptake, particlebombardment, agrotransformation, and the like. This process may resultin transient or stable expression of the transformed polynucleotide. By“stably transformed” is meant that the sequence of interest isintegrated into a replicon in the cell, such as a chromosome or episome.Transformed cells encompass not only the end product of a transformationprocess, but also the progeny thereof which retain the polynucleotide ofinterest.

For the purposes of the invention, “transgenic” refers to any cell,spore, tissue or part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

As used herein, the term “transposase” refers to an enzyme thatcatalyzes transposition. Preferred transposons are described in WO00/55346, PCT/US00/07317, and U.S. Ser. No. 09/658,859.

As used herein, the term “transposition” refers to a complex geneticrearrangement process involving the movement or copying of apolynucleotide (transposon) from one location and insertion intoanother, often within or between a genome or genomes, or DNA constructssuch as plasmids, bacmids, and cosmids.

As used herein, the term “transposon” (also known as a “transposableelement”, “transposable genetic element”, “mobile element”, or “jumpinggene”) refers to a mobile DNA element such as those, for example,described in WO 00/55346, PCT/US00/07317, and U.S. Ser. No. 09/658,859.Transposons can disrupt gene expression or cause deletions andinversions, and hence affect both the genotype and phenotype of theorganisms concerned. The mobility of transposable elements has long beenused in genetic manipulation, to introduce genes or other informationinto the genome of certain model systems.

As used herein, the term “Tween 20” means sorbitan mono-9-octadecenoatepoly(oxy-1,1-ethanediyl).

As used in this disclosure, the term “viability” of an organism refersto the ability of an organism to demonstrate growth under conditionsappropriate for said organism, or to demonstrate an active cellularfunction. Some examples of active cellular functions include respirationas measured by gas evolution, secretion of proteins and/or othercompounds, dye exclusion, mobility, dye oxidation, dye reduction,pigment production, changes in medium acidity, and the like.

The present inventors have discovered that disruption of the THR4 geneand/or gene product inhibits the pathogenicity of Magnaporthe grisea.Thus, the inventors are the first to demonstrate that Threonine synthaseis a target for antibiotics, preferably antifungals.

Accordingly, the invention provides methods for identifying compoundsthat inhibit THR4 gene expression or biological activity of its geneproduct(s). Such methods include ligand binding assays, assays forenzyme activity, cell-based assays, and assays for THR4 gene expression.Any compound that is a ligand for Threonine synthase may have antibioticactivity. For the purposes of the invention, “ligand” refers to amolecule that will bind to a site on a polypeptide. The compoundsidentified by the methods of the invention are useful as antibiotics.

Thus, in one embodiment, the invention provides a method for identifyinga test compound as a candidate for an antibiotic, comprising:

a) contacting a Threonine synthase polypeptide with a test compound; and

b) detecting the presence or absence of binding between said testcompound and said Threonine synthase polypeptide, wherein bindingindicates that said test compound is a candidate for an antibiotic.

The Threonine synthase protein may have the amino acid sequence of anaturally occurring Threonine synthase found in a fungus, animal, plant,or microorganism, or may have an amino acid sequence derived from anaturally occurring sequence. Preferably the Threonine synthase is afungal Threonine synthase. The cDNA (SEQ ID NO: 1) encoding theThreonine synthase protein, the genomic DNA (SEQ ID NO: 2) encoding theM. grisea protein, and the polypeptide (SEQ ID NO: 3) can be foundherein.

In one aspect, the invention also provides for a polypeptide consistingessentially of SEQ ID NO: 3. For the purposes of the invention, apolypeptide consisting essentially of SEQ ID NO: 3 has at least 80%sequence identity with SEQ ID NO: 3 and catalyses the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphatewith at least 10% of the activity of SEQ ID NO: 3. Preferably, thepolypeptide consisting essentially of SEQ ID NO: 3 has at least 85%sequence identity with SEQ ID NO: 3, more preferably the sequenceidentity is at least 90%, most preferably the sequence identity is atleast 95% or 97 or 99%, or any integer from 80-100% sequence identity inascending order. And, preferably, the polypeptide consisting essentiallyof SEQ ID NO: 3 has at least 25%, at least 50% , at least 75% or atleast 90% of the activity of M. grisea Threonine synthase, or anyinteger from 60-100% activity in ascending order.

By “fungal Threonine synthase” is meant an enzyme that can be found inat least one fungus, and which catalyzes the interconversion ofO-phospho-L-homoserine and water with L-threonine and orthophosphate.The Threonine synthase may be from any of the fungi, includingascomycota, zygomycota, basidiomycota, chytridiomycota, and lichens.

In one embodiment, the Threonine synthase is a Magnaporthe Threoninesynthase. Magnaporthe species include, but are not limited to,Magnaporthe rhizophila, Magnaporthe salvinii, Magnaporthe grisea andMagnaporthe poae and the imperfect states of Magnaporthe in the genusPyricularia. Preferably, the Magnaporthe Threonine synthase is fromMagnaporthe grisea.

In various embodiments, the Threonine synthase can be from Powdery Scab(Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot(Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot(Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporussquamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus(Armillaria gallica), Root rot (Armillaria luteobubalina), ShoestringRot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichummusae), Apple-rotting Fungus (Monilinia fructigena), Apple-rottingFungus (Penicillium expansum), Clubroot Disease (Plasmodiophorabrassicae), Potato Blight (Phytophthora infestans), Root pathogen(Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis),Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum), and thelike.

Fragments of a Threonine synthase polypeptide may be used in the methodsof the invention, preferably if the fragments include an intact ornearly intact epitope that occurs on the biologically active wildtypeThreonine synthase. The fragments comprise at least 10 consecutive aminoacids of a Threonine synthase. Preferably, the fragment comprises atleast 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, or at least540 consecutive amino acids residues of a Threonine synthase. In oneembodiment, the fragment is from a Magnaporthe Threonine synthase.Preferably, the fragment contains an amino acid sequence conserved amongfungal Threonine synthases.

Polypeptides having at least 50% sequence identity with a fungalThreonine synthase are also useful in the methods of the invention.Preferably, the sequence identity is at least 60%, more preferably thesequence identity is at least 70%, most preferably the sequence identityis at least 80% or 90 or 95 or 99%, or any integer from 60-100% sequenceidentity in ascending order.

In addition, it is preferred that the polypeptide has at least 10% ofthe activity of a fungal Threonine synthase. More preferably, thepolypeptide has at least 25%, at least 50%, at least 75% or at least 90%of the activity of a fungal Threonine synthase. Most preferably, thepolypeptide has at least 10%, at least 25%, at least 50%, at least 75%or at least 90% of the activity of the M. grisea Threonine synthaseprotein.

Thus, in another embodiment, the invention provides a method foridentifying a test compound as a candidate for a fungicide, comprising:

a) contacting a test compound with at least one polypeptide selectedfrom the group consisting of: a polypeptide having at least tenconsecutive amino acids of a fungal Threonine synthase; a polypeptidehaving at least 50% sequence identity with a fungal Threonine synthase;and a polypeptide having at least 10% of the activity of a fungalThreonine synthase; and

b) detecting the presence and/or absence of binding between said testcompound and said polypeptide, wherein binding indicates that said testcompound is a candidate for an antibiotic.

Any technique for detecting the binding of a ligand to its target may beused in the methods of the invention. For example, the ligand and targetare combined in a buffer. Many methods for detecting the binding of aligand to its target are known in the art, and include, but are notlimited to the detection of an immobilized ligand-target complex or thedetection of a change in the properties of a target when it is bound toa ligand. For example, in one embodiment, an array of immobilizedcandidate ligands is provided. The immobilized ligands are contactedwith a Threonine synthase protein or a fragment or variant thereof, theunbound protein is removed and the bound Threonine synthase is detected.In a preferred embodiment, bound Threonine synthase is detected using alabeled binding partner, such as a labeled antibody. In a variation ofthis assay, Threonine synthase is labeled prior to contacting theimmobilized candidate ligands. Preferred labels include fluorescent orradioactive moieties. Preferred detection methods include fluorescencecorrelation spectroscopy (FCS) and FCS-related confocal nanofluorimetricmethods.

Once a compound is identified as a candidate for an antibiotic, it canbe tested for the ability to inhibit Threonine synthase enzymaticactivity. The compounds can be tested using either in vitro or cellbased assays. Alternatively, a compound can be tested by applying itdirectly to a fungus or fungal cell, or expressing it therein, andmonitoring the fungus or fungal cell for changes or decreases in growth,development, viability, pathogenicity, or alterations in geneexpression. Thus, in one embodiment, the invention provides a method fordetermining whether a compound identified as an antibiotic candidate byan above method has antifungal activity, further comprising: contactinga fungus or fungal cells with said antifungal candidate and detecting adecrease in the growth, viability, or pathogenicity of said fungus orfungal cells.

By decrease in growth, is meant that the antifungal candidate causes atleast a 10% decrease in the growth of the fungus or fungal cells, ascompared to the growth of the fungus or fungal cells in the absence ofthe antifungal candidate. By a decrease in viability is meant that atleast 20% of the fungal cells, or portion of the fungus contacted withthe antifungal candidate are nonviable. Preferably, the growth orviability will be decreased by at least 40%. More preferably, the growthor viability will be decreased by at least 50%, 75% or at least 90% ormore. Methods for measuring fungal growth and cell viability are knownto those skilled in the art. By decrease in pathogenicity, is meant thatthe antifungal candidate causes at least a 10% decrease in the diseasecaused by contact of the fungal pathogen with its host, as compared tothe disease caused in the absence of the antifungal candidate.Preferably, the disease will be decreased by at least 40%. Morepreferably, the disease will be decreased by at least 50%, 75% or atleast 90% or more. Methods for measuring fungal disease are well knownto those skilled in the art, and include such metrics as lesionformation, lesion size, sporulation, respiratory failure, and/or death.

The ability of a compound to inhibit Threonine synthase activity can bedetected using in vitro enzymatic assays in which the disappearance of asubstrate or the appearance of a product is directly or indirectlydetected. Threonine synthase catalyzes the irreversible or reversiblereaction O-phospho-L-homoserine and water=L-threonine and orthophosphate(see FIG. 1). Methods for detection of O-phospho-L-homoserine,L-threonine, orthophosphate, and water, include spectrophotometry, massspectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a test compound asa candidate for an antibiotic, comprising:

a)contacting O-phospho-L-homoserine and water with a Threonine synthase;

b) contacting O-phospho-L-homoserine and water with Threonine synthaseand said test compound; and

c) determining the change in concentration for at least one of thefollowing: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a testcompound as a candidate for an antibiotic, comprising:

a) contacting L-threonine and orthophosphate with a Threonine synthase;

b) contacting L-threonine and orthophosphate with a Threonine synthaseand a test compound; and

c) determining the change in concentration for at least one of thefollowing: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

Enzymatically active fragments of a fungal Threonine synthase are alsouseful in the methods of the invention. For example, an enzymaticallyactive polypeptide comprising at least 100 consecutive amino acidresidues of a fungal Threonine synthase may be used in the methods ofthe invention. In addition, an enzymatically active polypeptide havingat least 50%, 60%, 70%, 80%, 90%, 95% or at least 98% sequence identitywith a fungal Threonine synthase may be used in the methods of theinvention. Most preferably, the polypeptide has at least 50% sequenceidentity with a fungal Threonine synthase and at least 10%, 25%, 75% orat least 90% of the activity thereof.

Thus, the invention provides a method for identifying a test compound asa candidate for an antibiotic, comprising:

a) contacting O-phospho-L-homoserine and water with a polypeptideselected from the group consisting of: a polypeptide having at least 50%sequence identity with a Threonine synthase, and a polypeptide having atleast 50% sequence identity with a Threonine synthase and having atleast 10% of the activity thereof, and a polypeptide comprising at least100 consecutive amino acids of a Threonine synthase;

b) contacting O-phospho-L-homoserine and water with said polypeptide anda test compound; and

c) determining the change in concentration for at least one of thefollowing: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

An additional method is provided by the invention for identifying a testcompound as a candidate for an antibiotic, comprising:

a) contacting L-threonine and orthophosphate with a polypeptide selectedfrom the group consisting of: a polypeptide having at least 50% sequenceidentity with a Threonine synthase, and a polypeptide having at least50% sequence identity with a Threonine synthase and at least 10% of theactivity thereof, and a polypeptide comprising at least 100 consecutiveamino acids of a Threonine synthase;

b) contacting L-threonine and orthophosphate, with said polypeptide anda test compound; and

c) determining the change in concentration for at least one of thefollowing, O-phospho-L-homoserine, L-threonine, orthophosphate, andwater, wherein a change in concentration for any of the above substancesindicates that said test compound is a candidate for an antibiotic.

For the in vitro enzymatic assays, Threonine synthase protein andderivatives thereof may be purified from a fungus or may berecombinantly produced in and purified from an archael, bacterial,fungal, or other eukaryotic cell culture. Preferably these proteins areproduced using an E. coli, yeast, or filamentous fungal expressionsystem. Methods for the purification of Threonine synthase may bedescribed in Malumbres et al. (1994) Appl Environ Microbiol 60: 2209-19(PMID: 8074505). Other methods for the purification of Threoninesynthase proteins and polypeptides are known to those skilled in theart.

As an alternative to in vitro assays, the invention also provides cellbased assays. In one embodiment, the invention provides a method foridentifying a test compound as a candidate for an antibiotic,comprising:

a) measuring the expression of a Threonine synthase in a cell, cells,tissue, or an organism in the absence of a test compound;

b) contacting said cell, cells, tissue, or organism with said testcompound and measuring the expression of said Threonine synthase in saidcell, cells, tissue, or organism; and

c) comparing the expression of Threonine synthase in steps (a) and (b),wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.

Expression of Threonine synthase can be measured by detecting the THR4primary transcript or mRNA, Threonine synthase polypeptide, or Threoninesynthase enzymatic activity. Methods for detecting the expression of RNAand proteins are known to those skilled in the art. See, for example,Current Protocols in Molecular Biology Ausubel et al., eds., GreenePublishing and Wiley-Interscience, New York, 1995. The method ofdetection is not critical to the invention. Methods for detecting THR4RNA include, but are not limited to amplification assays such asquantitative reverse transcriptase-PCR, and/or hybridization assays suchas Northern analysis, dot blots, slot blots, in-situ hybridization,transcriptional fusions using a THR4 promoter fused to a reporter gene,DNA assays, and microarray assays.

Methods for detecting protein expression include, but are not limitedto, immunodetection methods such as Western blots, ELISA assays,polyacrylamide gel electrophoresis, mass spectroscopy, and enzymaticassays. Also, any reporter gene system may be used to detect THR4protein expression. For detection using gene reporter systems, apolynucleotide encoding a reporter protein is fused in frame with THR4,so as to produce a chimeric polypeptide. Methods for using reportersystems are known to those skilled in the art.

Chemicals, compounds or compositions identified by the above methods asmodulators, preferably inhibitors, of THR4 expression or activity canthen be used to control fungal growth. Diseases such as rusts, mildews,and blights spread rapidly once established. Fungicides are thusroutinely applied to growing and stored crops as a preventive measure,generally as foliar sprays or seed dressings. For example, compoundsthat inhibit fungal growth can be applied to a fungus or expressed in afungus, in order to prevent fungal growth. Thus, the invention providesa method for inhibiting fungal growth, comprising contacting a funguswith a compound identified by the methods of the invention as havingantifungal activity.

Antifungals and antifungal inhibitor candidates identified by themethods of the invention can be used to control the growth of undesiredfungi, including ascomycota, zygomycota, basidiomycota, chytridiomycota,and lichens.

Examples of undesired fungi include, but are not limited to Powdery Scab(Spongospora subterranea), Grey Mould (Botrytis cinerea), White Rot(Armillaria mellea), Heartrot Fungus (Ganoderma adspersum), Brown-Rot(Piptoporus betulinus), Corn Smut (Ustilago maydis), Heartrot (Polyporussquamosus), Gray Leaf Spot (Cercospora zeae-maydis), Honey Fungus(Armillaria gallica), Root rot (Armillaria luteobubalina), ShoestringRot (Armillaria ostoyae), Banana Anthracnose Fungus (Colletotrichummusae), Apple-rotting Fungus (Monilinia fructigena), Apple-rottingFungus (Penicillium expansum), Clubroot Disease (Plasmodiophorabrassicae), Potato Blight (Phytophthora infestans), Root pathogen(Heterobasidion annosum), Take-all Fungus (Gaeumannomyces graminis),Dutch Elm Disease (Ophiostoma ulmi), Bean Rust (Uromycesappendiculatus), Northern Leaf Spot (Cochliobolus carbonum), MiloDisease (Periconia circinata), Southern Corn Blight (Cochliobolusheterostrophus), Leaf Spot (Cochliobolus lunata), Brown Stripe(Cochliobolus stenospilus), Panama disease (Fusarium oxysporum), WheatHead Scab Fungus (Fusarium graminearum), Cereal Foot Rot (Fusariumculmorum), Potato Black Scurf (Rhizoctonia solani), Wheat Black StemRust (Puccinia graminis), White mold (Sclerotinia sclerotiorum),diseases of animals such as infections of lungs, blood, brain, skin,scalp, nails or other tissues (Aspergillus fumigatus Aspergillus sp.Fusraium sp., Trichophyton sp., Epidermophyton sp., and Microsporum sp.,and the like).

Also provided is a method of screening for an antibiotic by determiningwhether a test compound is active against the gene identified (SEQ IDNO: 1 or SEQ ID NO: 2), its gene product (SEQ ID NO: 3), or thebiochemical pathway or pathways on which it functions.

In one particular embodiment, the method is performed by providing anorganism having a first form of the gene corresponding to either SEQ IDNO: 1 or SEQ ID NO: 2, either a normal form, a mutant form, a homologue,or a heterologous THR4 gene that performs a similar function as THR4.The first form of THR4 may or may not confer a growth conditionalphenotype, i.e., a L-threonine requiring phenotype, and/or ahypersensitivity or hyposensitivity phenotype on the organism havingthat altered form. In one particular embodiment a mutant form contains atransposon insertion. A comparison organism having a second form of aTHR4, different from the first form of the gene is also provided, andthe two organisms are separately contacted with a test compound. Thegrowth of the two organisms in the presence of the test compound is thencompared.

Thus, in one embodiment, the invention provides a method for identifyinga test compound as a candidate for an antibiotic, comprising:

a) providing cells having one form of a Threonine synthase gene, andproviding comparison cells having a different form of a Threoninesynthase gene; and

b) contacting said cells and said comparison cells with a test compoundand determining the growth of said cells and said comparison cells inthe presence of the test compound, wherein a difference in growthbetween said cells and said comparison cells in the presence of saidtest compound indicates that said test compound is a candidate for anantibiotic.

It is recognized in the art that the optional determination of thegrowth of said first organism and said comparison second organism in theabsence of any test compounds may be performed to control for anyinherent differences in growth as a result of the different genes. It isalso recognized that any combination of two different forms of a THR4gene, including normal genes, mutant genes, homologues, and functionalhomologues may be used in this method. Growth and/or proliferation of anorganism is measured by methods well known in the art such as opticaldensity measurements, and the like. In a preferred embodiment theorganism is Magnaporthe grisea.

Conditional lethal mutants may identify particular biochemical and/orgenetic pathways given that at least one identified target gene ispresent in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics as inhibitorsof the substrates, products and enzymes of the pathway. Pathways knownin the art may be found at the Kyoto Encyclopedia of Genes and Genomesand in standard biochemistry texts (Lehninger, A., D. Nelson, et al.(1993) Principles of Biochemistry, New York, Worth Publishers).

Thus, in one embodiment, the invention provides a method for screeningfor test compounds acting against the biochemical and/or genetic pathwayor pathways in which THR4 functions, comprising:

a) providing cells having one form of a gene in the L-threoninebiochemical and/or genetic pathway and providing comparison cells havinga different form of said gene;

b) contacting said cells and said comparison cells with a test compound;and

c) determining the growth of said cells and said comparison cells in thepresence of said test compound, wherein a difference in growth betweensaid cells and said comparison cells in the presence of said testcompound indicates that said test compound is a candidate for anantibiotic.

The use of multi-well plates for screening is a format that readilyaccommodates multiple different assays to characterize variouscompounds, concentrations of compounds, and fungal strains in varyingcombinations and formats. Certain testing parameters for the screeningmethod can significantly affect the identification of growth inhibitors,and thus can be manipulated to optimize screening efficiency and/orreliability. Notable among these factors are variable sensitivities ofdifferent mutants, increasing hypersensitivity with increasingly lesspermissive conditions, an apparent increase in hypersensitivity withincreasing compound concentration, and other factors known to those inthe art.

Conditional lethal mutants may identify particular biochemical and/orgenetic pathways given that at least one identified target gene ispresent in that pathway. Knowledge of these pathways allows for thescreening of test compounds as candidates for antibiotics. Pathwaysknown in the art may be found at the Kyoto Encyclopedia of Genes andGenomes and in standard biochemistry texts (Lehninger et al. (1993)Principles of Biochemistry).

Thus, in one embodiment, the invention provides a method for screeningfor test compounds acting against the biochemical and/or genetic pathwayor pathways in which THR4 functions, comprising:

(a) providing paired growth media comprising a first medium and a secondmedium, wherein said second medium contains a higher level ofL-threonine than said first medium;

(b) contacting an organism with a test compound;

(c) inoculating said first and said second media with said organism; and

(d) determining the growth of said organism, wherein a difference ingrowth of the organism between said first and said second mediaindicates that said test compound is a candidate for an antibiotic.

It is recognized in the art that determination of the growth of saidorganism in the paired media in the absence of any test compounds may beperformed to control for any inherent differences in growth as a resultof the different media. Growth and/or proliferation of an organism ismeasured by methods well known in the art such as optical densitymeasurements, and the like. In a preferred embodiment, the organism isMagnaporthe grisea.

EXPERIMENTAL EXAMPLE 1 Construction of Plasmids with a TransposonContaining a Selectable Marker

Construction of Sif transposon

Sif was constructed using the GPS3 vector from the GPS-M mutagenesissystem from New England Biolabs, Inc. (Beverly, Mass.) as a backbone.This system is based on the bacterial transposon Tn7. The followingmanipulations were done to GPS3 according to Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress. The kanamycin resistance gene (npt) contained between the Tn7arms was removed by EcoRV digestion. The bacterial hygromycin Bphosphotransferase (hph) gene (Gritz and Davies (1983) Gene 25: 179-88(PMID: 6319235)) under control of the Aspergillus nidulans trpC promoterand terminator (Mullaney et al. (1985) Mol Gen Genet 199: 37-45 (PMID:3158796)) was cloned by a HpaI/EcoRV blunt ligation into the Tn7 arms ofthe GPS3 vector yielding pSifl. Excision of the ampicillin resistancegene (bla) from pSifl was achieved by cutting pSifl with XmnI and BglIfollowed by a T4 DNA polymerase treatment to remove the 3′ overhangsleft by the BglI digestion and religation of the plasmid to yield pSif.Top 10F′ electrocompetent E. coli cells (Invitrogen) were transformedwith ligation mixture according to manufacturer'srecommendations.Transformants containing the Sif transposon wereselected on LB agar (Sambrook et al. (1989) Molecular Cloning, aLaboratory Manual) containing 50 ug/ml of hygromycin B (Sigma Chem. Co.,St. Louis, Mo.).

EXAMPLE 2 Construction of a Fungal Cosmid Library

Cosmid libraries were constructed in the pcosKA5 vector (Hamer et al.(2001) Proc Natl Acad Sci USA 98: 5110-15 (PMID: 11296265)) as describedin Sambrook et al. (1989) Molecular Cloning, a Laboratory Manual. Cosmidlibraries were quality checked by pulsed-field gel electrophoresis,restriction digestion analysis, and PCR identification of single genes.

EXAMPLE 3 Construction of Cosmids with Transposon Insertion into FungalGenes

Sif Transposition into a Cosmid

Transposition of Sif into the cosmid framework was carried out asdescribed by the GPS-M mutagenesis system (New England Biolabs, Inc.).Briefly, 2 ul of the 10×GPS buffer, 70 ng of supercoiled pSIF, 8-12 ugof target cosmid DNA were mixed and taken to a final volume of 20 ulwith water. 1 ul of transposase (TnsABC) was added to the assemblyreaction and incubated for 10 minutes at 37° C. After the assemblyreaction, 1 ul of start solution was added to the tube, mixed well andincubated for 1 hour at 37° C. followed by heat inactivation of theproteins at 75° C. for 10 min. Destruction of the remaining untransposedpSif was done by PISceI digestion at 37° C. for 2 hours followed by 10min incubation at 75° C. to inactivate the proteins. Transformation ofTop10F′ electrocompetent cells (Invitrogen) was done according tomanufacturers recommendations. Sif-containing cosmid transformants wereselected by growth on LB agar plates containing 50 ug/ml of hygromycin B(Sigma Chem. Co.) and 100 ug/ml of Ampicillin (Sigma Chem. Co.).

EXAMPLE 4 High Throughput Preparation and Verification of TransposonInsertion into the M. grisea THR4 Gene

E. coli strains containing cosmids with transposon insertions werepicked to 96 well growth blocks (Beckman Co.) containing 1.5 ml of TB(Terrific Broth, Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press) supplemented with 50 ug/mlof ampicillin. Blocks were incubated with shaking at 37° C. overnight.E. coli cells were pelleted by centrifugation and cosmids were isolatedby a modified alkaline lysis method (Marra et al. (1997) Genome Res 7:1072-84 (PMID: 9371743)). DNA quality was checked by electrophoresis onagarose gels. Cosmids were sequenced using primers from the ends of eachtransposon and commercial dideoxy sequencing kits (Big Dye Terminators,Perkin Elmer Co.). Sequencing reactions were analyzed on an ABI377 DNAsequencer (Perkin Elmer Co.).

DNA sequences adjacent to the site of the insertion were collected andused to search DNA and protein databases using the BLAST algorithms(Altschul et al. (1997) Nucleic Acids Res 25: 3389-3402 (PMID:9254694)). A single insertion of SIF into the Magnaporthe grisea THR4gene was chosen for further analysis. This construct was designatedcpgmra0012020a04 and it contains the SIF transposon approximatelybetween amino acids 314 and 315 relative to the Schizosaccharomycespombe homologue ThrC (total length: 514 amino acids, GENBANK: 2501152).

EXAMPLE 5 Preparation of THR4 Cosmid DNA and Transformation ofMagnaporthe grisea

Cosmid DNA from the THR4 transposon tagged cosmid clone was preparedusing QIAGEN Plasmid Maxi Kit (QIAGEN), and digested by PI-Pspl (NewEngland Biolabs, Inc.). Fungal electro-transformation was performedessentially as described (Wu et al. (1997) MPMI 10: 700-708). Briefly,M. grisea strain Guy 11 was grown in complete liquid media (Talbot etal. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) shaking at 120 rpmfor 3 days at 25° C. in the dark. Mycelia was harvested and washed withsterile H₂O and digested with 4 mg/ml beta-glucanase (InterSpex) for 4-6hours to generate protoplasts. Protoplasts were collected bycentrifugation and resuspended in 20% sucrose at the concentration of2×10⁸ protoplasts/ml. 50 ul protoplast suspension was mixed with 10-20ug of the cosmid DNA and pulsed using Gene Pulser II (BioRad) set withthe following parameters: resistance 200 ohm, capacitance 25 uF, voltage0.6 kV. Transformed protoplasts were regenerated in complete agar media(CM, Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) withthe addition of 20% sucrose for one day, then overlayed with CM agarmedia containing hygromycin B (250 ug/ml) to select transformants.Transformants were screened for homologous recombination events in thetarget gene by PCR (Hamer et al. (2001) Proc Natl Acad Sci USA 98:5110-15 (PMID: 11296265)). Two independent strains were identified andare hereby referred to as KO1-3 and KO1-22, respectively.

EXAMPLE 6 Effect of Transposon Insertion on Magnaporthe Pathogenicity

The target fungal strains, KO1-3 and KO1-22, obtained in Example 5 andthe wild type strain, Guy11, were subjected to a pathogenicity assay toobserve infection over a 1-week period. Rice infection assays wereperformed using Indian rice cultivar CO39 essentially as described inValent et al. ((1991) Genetics 127: 87-101 (PMID: 2016048)). All threestrains were grown for spore production on complete agar media. Sporeswere harvested and the concentration of spores adjusted for whole plantinoculations. Two-week-old seedlings of cultivar CO39 were sprayed with12 ml of conidial suspension (5×10⁴ conidia per ml in 0.01% Tween-20(Polyoxyethylensorbitan monolaureate) solution). The inoculated plantswere incubated in a dew chamber at 27° C. in the dark for 36 hours, andtransferred to a growth chamber (27° C. 12 hours/21° C. 12 hours 70%humidity) for an additional 5.5 days. Leaf samples were taken at 3, 5,and 7 days post-inoculation and examined for signs of successfulinfection (i.e. lesions). FIG. 2 shows the effects of THR4 genedisruption on Magnaporthe infection at five days post-inoculation.

EXAMPLE 7 Verification of THR4 Gene Function by Analysis of NutritionalRequirements

The fungal strains, KO1-3 and KO1-22, containing the THR4 disrupted geneobtained in Example 5 were analyzed for their nutritional requirementfor L-threonine using the PM5 phenotype microarray from Biolog, Inc.(Hayward, Calif.). The PM5 plate tests for the auxotrophic requirementfor 94 different metabolites. The inoculating fluid consists of 0.05%Phytagel, 0.03% Pluronic F68, 1% glucose, 23.5 mM NaNO₃, 6.7 mM KCl, 3.5mM Na₂SO₄, 11 mM KH₂PO₄, 0.01% p-iodonitrotetrazolium violet, 0.1 mMMgCl₂, 1.0 mM CaCl₂ and trace elements, pH adjusted to 6.0 with NaOH.Final concentrations of trace elements are: 7.6 μM ZnCl₂, 2.5 μMMnCl₂.4H₂O, 1.8 μM FeCl₂.4H₂O, 0.71 μM CoCl₂.6H₂O, 0.64 μM CuCl₂.2H₂O,0.62 μM Na₂MoO₄, 18 μM H₃BO₃. Spores for each strain were harvested intothe inoculating fluid. The spore concentrations were adjusted to 2×10⁵spores/ml. 100 μl of spore suspension were deposited into each well ofthe microtiter plates. The plates were incubated at 25° C. for 7 days.Optical density (OD) measurements at 490 nm and 750 nm were taken daily.The OD₄₉₀ measures the extent of tetrazolium dye reduction and the levelof growth, and OD₇₅₀ measures growth only. Turbidity=OD₄₉₀+OD₇₅₀. Dataconfirming the annotated gene function is presented as a graph ofTurbidity vs. Time showing both the mutant fungi and the wild-typecontrol in the absence (FIG. 3A) and presence (FIG. 3B) of L-threonine.

EXAMPLE 8 Cloning and Expression Strategies, Extraction and Purificationof Threonine Synthase Protein

The following protocol may be employed to obtain a purified Threoninesynthase protein.

Cloning and Expression Strategies

A THR4 cDNA gene can be cloned into E. coli (pET vectors-Novagen),Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectorscontaining His/fusion protein tags, and the expression of recombinantprotein can be evaluated by SDS-PAGE and Western blot analysis.

Extraction

Extract recombinant protein from 250 ml cell pellet in 3 ml ofextraction buffer by sonicating 6 times, with 6 sec pulses at 4° C.Centrifuge extract at 15000×g for 10 min and collect supernatant. Assessbiological activity of the recombinant protein by activity assay.

Purification

Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen).Purification protocol: perform all steps at 4° C.:

Use 3 ml Ni-beads (Qiagen)

Equilibrate column with the buffer

Load protein extract

Wash with the equilibration buffer

Elute bound protein with 0.5 M imidazole

EXAMPLE 9 Assays for Testing Binding of Test Compounds to ThreonineSynthase

The following protocol may be employed to identify test compounds thatbind to the Threonine synthase protein.

Purified full-length Threonine synthase polypeptide with a His/fusionprotein tag (Example 8) is bound to a HisGrab™ Nickel Coated Plate(Pierce, Rockford, Ill.). following manufacturer's instructions.

Buffer conditions are optimized (e.g. ionic strength or pH, Ramos andCalderon (1994) FEBS Lett 351: 357-9 (PMID: 8082795)) for binding ofradiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia 59:1238-44 (PMID: 7819407)) to the bound Threonine synthase.

Screening of test compounds is performed by adding test compound andradiolabeled O-phospho-L-homoserine (Gening et al. (1994) Biokhimiia 59:1238-44 (PMID: 7819407)) to the wells of the HisGrab™ plate containingbound Threonine synthase.

The wells are washed to remove excess labeled ligand and scintillationfluid (Scintiverse®, Fisher Scientific) is added to each well.

The plates are read in a microplate scintillation counter.

Candidate compounds are identified as wells with lower radioactivity ascompared to control wells with no test compound added.

Additionally, a purified polypeptide comprising 10-50 amino acids fromthe M. grisea Threonine synthase is screened in the same way. Apolypeptide comprising 10-50 amino acids is generated by subcloning aportion of the THR4 gene into a protein expression vector that adds aHis-Tag when expressed (see Example 8). Oligonucleotide primers aredesigned to amplify a portion of the THR4 gene using the polymerasechain reaction amplification method. The DNA fragment encoding apolypeptide of 10-50 amino acids is cloned into an expression vector,expressed in a host organism and purified as described in Example 8above.

Test compounds that bind THR4 are further tested for antibioticactivity. M. grisea is grown as described for spore production onoatmeal agar media (Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID:8312740)). Spores are harvested into minimal media (Talbot et al. (1993)Plant Cell 5: 1575-1590 (PMID: 8312740)) to a concentration of 2×10⁵spores/ml and the culture is divided. The test compound is added to oneculture to a final concentration of 20-100 μg/ml. Solvent only is addedto the second culture. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The growthcurves of the solvent control sample and the test compound sample arecompared. A test compound is an antibiotic candidate if the growth ofthe culture containing the test compound is less than the growth of thecontrol culture.

EXAMPLE 10 Assays for Testing Inhibitors or Candidates for Inhibition ofThreonine Synthase Activity

The enzymatic activity of Threonine synthase is determined in thepresence and absence of candidate compounds in a suitable reactionmixture, such as described by Ramos and Calderon (1994) FEBS Lett 351:357-9 (PMID: 8082795). Candidate compounds are identified when adecrease in products or a lack of decrease in substrates is detectedwith the reaction proceeding in either direction.

Additionally, the enzymatic activity of a polypeptide comprising 10-50amino acids from the M. grisea Threonine synthase is determined in thepresence and absence of candidate compounds in a suitable reactionmixture, such as described by Ramos and Calderon (1994) FEBS Lett 351:357-9 (PMID: 8082795). A polypeptide comprising 10-50 amino acids isgenerated by subcloning a portion of the THR4 gene into a proteinexpression vector that adds a His-Tag when expressed (see Example 8).Oligonucleotide primers are designed to amplify a portion of the THR4gene using polymerase chain reaction amplification method. The DNAfragment encoding a polypeptide of 10-50 amino acids is cloned into anexpression vector, expressed and purified as described in Example 8above.

Test compounds identified as inhibitors of THR4 activity are furthertested for antibiotic activity. Magnaporthe grisea fungal cells aregrown under standard fungal growth conditions that are well known anddescribed in the art. M. grisea is grown as described for sporeproduction on oatmeal agar media (Talbot et al. (1993) Plant Cell 5:1575-1590 (PMID: 8312740)). Spores are harvested into minimal media(Talbot et al. (1993) Plant Cell 5: 1575-1590 (PMID: 8312740)) to aconcentration of 2×10⁵ spores/ml and the culture is divided. The testcompound is added to one culture to a final concentration of 20-100μg/ml. Solvent only is added to the second culture. The plates areincubated at 25° C. for seven days and optical density measurements at590 nm are taken daily. The growth curves of the solvent control sampleand the test compound sample are compared. A test compound is anantibiotic candidate if the growth of the culture containing the testcompound is less than the growth of the control culture.

EXAMPLE 11 Assays for Testing Compounds for Alteration of ThreonineSynthase Gene Expression

Magnaporthe grisea fungal cells are grown under standard fungal growthconditions that are well known and described in the art. Wild-type M.grisea spores are harvested from cultures grown on complete agar oroatmeal agar media after growth for 10-13 days in the light at 25° C.using a moistened cotton swab. The concentration of spores is determinedusing a hemacytometer and spore suspensions are prepared in a minimalgrowth medium to a concentration of 2×10⁵ spores per ml. 25 ml culturesare prepared to which test compounds will be added at variousconcentrations. A culture with no test compound present is included as acontrol. The cultures are incubated at 25° C. for 3 days after whichtest compound or solvent only control is added. The cultures areincubated an additional 18 hours. Fungal mycelia is harvested byfiltration through Miracloth (CalBiochem®, La Jolla, Calif.), washedwith water and frozen in liquid nitrogen. Total RNA is extracted withTRIZOL® Reagent using the methods provided by the manufacturer (LifeTechnologies, Rockville, Md.). Expression is analyzed by Northernanalysis of the RNA samples as described (Sambrook et al. (1989)Molecular Cloning, a Laboratory Manual, Cold Spring Harbor LaboratoryPress) using a radiolabeled fragment of the THR4 gene as a probe. Testcompounds resulting in a reduced level of THR4 mRNA relative to theuntreated control sample are identified as candidate antibioticcompounds.

EXAMPLE 12 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Threonine Synthase with No Activity

Magnaporthe grisea fungal cells containing a mutant form of the THR4gene which abolishes enzyme activity, such as a gene containing atransposon insertion (see Examples 4 and 5), are grown under standardfungal growth conditions that are well known and described in the art.Magnaporthe grisea spores are harvested from cultures grown on completeagar medium containing 4 mM L-threonine (Sigma-Aldrich Co.) after growthfor 10-13 days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium containing 100 μML-threonine to a concentration of 2×10⁵ spores per ml. Approximately4×10⁴ spores are added to each well of 96-well plates to which a testcompound is added (at varying concentrations). The total volume in eachwell is 200 μl. Wells with no test compound present (growth control),and wells without cells are included as controls (negative control). Theplates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221 (PMID: 7749303)).

EXAMPLE 13 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of Threonine Synthase with ReducedActivity

Magnaporthe grisea fungal cells containing a mutant form of the THR4gene, such as a promoter truncation that reduces expression, are grownunder standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea sporesare harvested from cultures grown on complete agar medium containing 4mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in thelight at 25° C. using a moistened cotton swab. The concentration ofspores is determined using a hemacytometer and spore suspensions areprepared in a minimal growth medium to a concentration of 2×10⁵ sporesper ml. Approximately 4×10⁴ spores are added to each well of 96-wellplates to which a test compound is added (at varying concentrations).The total volume in each well is 200 μl. Wells with no test compoundpresent (growth control), and wells without cells are included ascontrols (negative control). The plates are incubated at 25° C. forseven days and optical density measurements at 590 nm are taken daily.Wild type cells are screened under the same conditions. The effect ofeach compound on the mutant and wild-type fungal strains is measuredagainst the growth control and the percent of inhibition is calculatedas the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on a fungal strain and that on the wild-type cells arecompared. Compounds that show differential growth inhibition between themutant and the wild type are identified as potential antifungalcompounds. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221).

EXAMPLE 14 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-threonine Biosynthetic Gene withNo Activity

Magnaporthe grisea fungal cells containing a mutant form of a gene inthe L-threonine biosynthetic pathway (e.g. Homoserine kinase (E.C.2.7.1.39)) are grown under standard fungal growth conditions that arewell known and described in the art. Magnaporthe grisea spores areharvested from cultures grown on complete agar medium containing 4 mML-threonine (Sigma-Aldrich Co.) after growth for 10-13 days in the lightat 25° C. using a moistened cotton swab. The concentration of spores isdetermined using a hemacytometer and spore suspensions are prepared in aminimal growth medium containing 100 μM L-threonine to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild-type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221).

EXAMPLE 15 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Mutant Form of a L-threonine Biosynthetic Gene withReduced Activity

Magnaporthe grisea fungal cells containing a mutant form of a gene inthe L-threonine biosynthetic pathway (e.g. Homoserine kinase (E.C.2.7.1.39)), such as a promoter truncation that reduces expression, aregrown under standard fungal growth conditions that are well known anddescribed in the art. A promoter truncation is made by deleting aportion of the promoter upstream of the transcription start site usingstandard molecular biology techniques that are well known and describedin the art (Sambrook et al. (1989) Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory Press). Magnaporthe grisea fungalcells containing a mutant form of are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumcontaining 4 mM L-threonine (Sigma-Aldrich Co.) after growth for 10-13days in the light at 25° C. using a moistened cotton swab. Theconcentration of spores is determined using a hemacytometer and sporesuspensions are prepared in a minimal growth medium to a concentrationof 2×10⁵ spores per ml. Approximately 4×10⁴ spores or cells areharvested and added to each well of 96-well plates to which growth mediais added in addition to an amount of test compound (at varyingconcentrations). The total volume in each well is 200 μl. Wells with notest compound present, and wells without cells are included as controls.The plates are incubated at 25° C. for seven days and optical densitymeasurements at 590 nm are taken daily. Wild type cells are screenedunder the same conditions. The effect of each compound on the mutant andwild-type fungal strains is measured against the growth control and thepercent of inhibition is calculated as the OD₅₉₀ (fungal strain plustest compound)/OD₅₉₀ (growth control)×100. The percent of growthinhibition as a result of a test compound on a fungal strain and that onthe wild type cells are compared. Compounds that show differentialgrowth inhibition between the mutant and the wild type are identified aspotential antifungal compounds. Similar protocols may be found in Kirschand DiDomenico ((1994) Biotechnology 26: 177-221).

EXAMPLE 16 In Vivo Cell Based Assay Screening Protocol with a FungalStrain Containing a Fungal THR4 and a Second Fungal Strain Containing aHeterologous THR4 Gene

Wild-type Magnaporthe grisea fungal cells and M. grisea fungal cellslacking a functional THR4 gene and containing a Thr4 gene fromSaccharomyces cerevisiae (Genbank: 6319901, 50% sequence identity) aregrown under standard fungal growth conditions that are well known anddescribed in the art. A M. grisea strain carrying a heterologous THR4gene is made as follows:

A M. grisea strain is made with a nonfunctional THR4 gene, such as onecontaining a transposon insertion in the native gene (see Examples 4 and5).

A construct containing a heterologous THR4 gene is made by cloning theThr4 gene from Saccharomyces cerevisiae into a fungal expression vectorcontaining a trpC promoter and terminator (e.g. pCB1003, Carroll et al.(1994) Fungal Gen News Lett 41: 22) using standard molecular biologytechniques that are well known and described in the art (Sambrook et al.(1989) Molecular Cloning, a Laboratory Manual).

The said construct is used to transform the M. grisea strain lacking afunctional THR4 gene (see Example 5). Transformants are selected onminimal agar medium lacking L-threonine. Only transformants carrying afunctional THR4 gene will grow.

Wild-type strains of Magnaporthe grisea and strains containing aheterologous form of THR4 are grown under standard fungal growthconditions that are well known and described in the art. Magnaporthegrisea spores are harvested from cultures grown on complete agar mediumafter growth for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium to a concentration of 2×10⁵ spores per ml. Approximately 4×10⁴spores or cells are harvested and added to each well of 96-well platesto which growth media is added in addition to an amount of test compound(at varying concentrations). The total volume in each well is 200 μl.Wells with no test compound present, and wells without cells areincluded as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. The effectof each compound on the wild-type and heterologous fungal strains ismeasured against the growth control and the percent of inhibition iscalculated as the OD₅₉₀ (fungal strain plus test compound)/OD₅₉₀ (growthcontrol)×100. The percent of growth inhibition as a result of a testcompound on the wild-type and heterologous fungal strains are compared.Compounds that show differential growth inhibition between the wild-typeand heterologous strains are identified as potential antifungalcompounds with specificity to the native or heterologous THR4 geneproducts. Similar protocols may be found in Kirsch and DiDomenico((1994) Biotechnology 26: 177-221).

EXAMPLE 17 Pathway Specific In Vivo Assay Screening Protocol

Magnaporthe grisea fungal cells are grown under standard fungal growthconditions that are well known and described in the art. Wild-type M.grisea spores are harvested from cultures grown on oatmeal agar mediaafter growth. for 10-13 days in the light at 25° C. using a moistenedcotton swab. The concentration of spores is determined using ahemacytometer and spore suspensions are prepared in a minimal growthmedium and a minimal growth medium containing 4 mM L-threonine(Sigma-Aldrich Co.) to a concentration of 2×10⁵ spores per ml. Theminimal growth media contains carbon, nitrogen, phosphate, and sulfatesources, and magnesium, calcium, and trace elements (for example, seeinoculating fluid in Example 7). Spore suspensions are added to eachwell of a 96-well microtiter plate (approximately 4×10⁴ spores/well).For each well containing a spore suspension in minimal media, anadditional well is present containing a spore suspension in minimalmedium containing 4 mM L-threonine. Test compounds are added to wellscontaining spores in minimal media and minimal media containingL-threonine. The total volume in each well is 200 μl. Both minimal mediaand L-threonine containing media wells with no test compound areprovided as controls. The plates are incubated at 25° C. for seven daysand optical density measurements at 590 nm are taken daily. A compoundis identified as a candidate for an antibiotic acting against theL-threonine biosynthetic pathway when the observed growth in the wellcontaining minimal media is less than the observed growth in the wellcontaining L-threonine as a result of the addition of the test compound.Similar protocols may be found in Kirsch and DiDomenico ((1994)Biotechnology 26: 177-221).

While the foregoing describes certain embodiments of the invention, itwill be understood by those skilled in the art that variations andmodifications may be made and still fall within the scope of theinvention. The foregoing examples are intended to exemplify variousspecific embodiments of the invention and do not limit its scope in anymanner.

3 1 1650 DNA Magnaporthe grisea 1 atggagaacg gtgctgcaac caacggggcgtcggagaagt cgcactctcc ttcacagacc 60 tacctctcca caaggggaga cgattatgggctctcattcg agaccgtcgt cctcaaaggt 120 cttgcggctg acgggggtct tttcctgcccgaggaagtgc ccgcggcaac cgagtggcaa 180 agctggaaag acctgcccta caccgagcttgccgtcaagg ttctcagctt gtacatctcc 240 cccgccgagg tgccgacgga agacctcagggcgctcgtcg agcgcagcta ctcgaccttc 300 cgatccaagg aggttgtgcc gctggtgaagctggaggaca accttcacct gctggagcta 360 ttccacggcc ccagctactc gttcaaggactgcgcgctgc aattccttgg taacctcttc 420 gagtactttt tgactcgcaa gaacaagggaaaggagggca aagacaggca ccacctcact 480 gtggtcggcg caacaagtgg tgataccggttcggcggcca tctatggtct tcgcaacaag 540 aaggatgttt ccgtcttcat cctgcaccccaagggtcgtg taagccccat ccaggaggcc 600 cagatgacca cggtgctcga ccaaaatgttcacaaccttg ccgtgaccgg cacctttgac 660 gattgccaag atatcgtcaa ggccatgttcaacgacccag attcgaatgc gacactgaag 720 cttggtgctg tcaactcgat caactggtccaggatattgg cccagattgt ttactacttc 780 cactcgtact tttctctggc cagggcgtcaccagagacgt tcaaggtcgg cgacaaagtc 840 cgctttgtca cccccaccgg gaactttggtaacatcctgg ctggatactt tgcacaaaag 900 atgggcttgc ctgtcgacaa gttggtcgttgcgacaaatg agaacgacat tcttgacagg 960 ttttggaaga cgggccgcta cgaaaagaagcctgcaagcc ccgaggaagc cgcaggcggt 1020 ctgcctcaag atggcgtaaa ggctcacgaggagggctgca aggagaccct gagcccggcg 1080 atggacattt tggtgtcgag caactttgagcgaacactgt ggtttcttgc caaggagttc 1140 gctgctacgg ctggcctcaa tgacgagttcaacaagaagc aagccggcca ggaagttgtg 1200 gcatggtaca agtccctcaa ggctaccggaggcttcggtc cggtccaccc tgaaatcatg 1260 gacaatggcc gccaggtctt tgaaagcgagcgcgtgagcg acacccagac cctcgagatg 1320 atcgcggaga tgtacaaagc cacaaagtacgttctcgacc cgcactctgc cgtcggtgtt 1380 gcgggggcca agaggtcaat gtcgagggcctccaacgtcc cgcacatcgc gctgtccacg 1440 gcccacccag ccaagttctc tggcgccgttgagcttgcgc tcaaggacca gaaggagttc 1500 gactttacaa agcaggtcct gccagaggactttgttggac tagcagagaa ggaaaagagg 1560 gtgactgagg tggccgcgaa ctggcaggaagtgagggaga ttgtcaagaa gcaggtcgag 1620 gaagacttga aggctgaaag tagtgcataa1650 2 2082 DNA Magnaporthe grisea 2 tacgctgtca aataggcgat ggccgattacctattttgta ttgacaaaaa atgacaagac 60 cagctgtatc cactgatatc gataaggttttttattactg gccgatgtcg ggagacgcgg 120 ggcgaggtgg gcgaaattga ctaacactgattttgactga tgcgactgat gcgacagccg 180 cgcgacaaca cccaacacgc agacttgacagattctgcta ctacaaatcc tgcatattta 240 acagcgctgc aactcgacga tggagaacggtgctgcaacc aacggggcgt cggagaagtc 300 gcactctcct tcacagacct acctctccacaaggggagac gattatgggc tctcattcga 360 gaccgtcgtc ctcaaaggtc ttgcggctgacgggggtctt ttcctgcccg aggaagtgcc 420 cgcggcaacc gagtggcaaa gctggaaagacctgccctac accgagcttg ccgtcaaggt 480 tctcagcttg tacatctccc ccgccgaggtgccgacggaa gacctcaggg cgctcgtcga 540 gcgcagctac tcgaccttcc gatccaaggaggttgtgccg ctggtgaagc tggaggacaa 600 ccttcacctg ctggagctat tccacggccccagctactcg ttcaaggact gcgcgctgca 660 attccttggt aacctcttcg agtactttttgactcgcaag aacaagggaa aggagggcaa 720 agacaggcac cacctcactg tggtcggcgcaacaagtggt gataccggtt cggcggccat 780 ctatggtctt cgcaacaaga aggatgtttccgtcttcatc ctgcacccca agggtcgtgt 840 aagccccatc caggaggccc agatgaccacggtgctcgac caaaatgttc acaaccttgc 900 cgtgaccggc acctttgacg attgccaagatatcgtcaag gccatgttca acgacccaga 960 ttcgaatgcg acactgaagc ttggtgctgtcaactcgatc aactggtcca ggatattggc 1020 ccagattgtt tactacttcc actcgtacttttctctggcc agggcgtcac cagagacgtt 1080 caaggtcggc gacaaagtcc gctttgtcacccccaccggg aactttggta acatcctggc 1140 tggatacttt gcacaaaaga tgggcttgcctgtcgacaag ttggtcgttg cgacaaatga 1200 gaacgacatt cttgacaggt tttggaagacgggccgctac gaaaagaagc ctgcaagccc 1260 cgaggaagcc gcaggcggtc tgcctcaagatggcgtaaag gctcacgagg agggctgcaa 1320 ggagaccctg agcccggcga tggacattttggtgtcgagc aactttgagc gaacactgtg 1380 gtttcttgcc aaggagttcg ctgctacggctggcctcaat gacgagttca acaagaagca 1440 agccggccag gaagttgtgg catggtacaagtccctcaag gctaccggag gcttcggtcc 1500 ggtccaccct gaaatcatgg acaatggccgccaggtcttt gaaagcgagc gcgtgagcga 1560 cacccagacc ctcgagatga tcgcggagatgtacaaagcc acaaagtacg ttctcgaccc 1620 gcactctgcc gtcggtgttg cgggggccaagaggtcaatg tcgagggcct ccaacgtccc 1680 gcacatcgcg ctgtccacgg cccacccagccaagttctct ggcgccgttg agcttgcgct 1740 caaggaccag aaggagttcg actttacaaagcaggtcctg ccagaggact ttgttggact 1800 agcagagaag gaaaagaggg tgactgaggtggccgcgaac tggcaggaag tgagggagat 1860 tgtcaagaag caggtcgagg aagacttgaaggctgaaagt agtgcataat cacgagccgg 1920 agtgcagtag aaaatggtgt cgagatcagcatctagattt gctttcctag agatatgcaa 1980 acatttactt attctggacc ctgaatgcagccccaagggt gcactagatc ggataactgg 2040 aggtttagac gcggccgact tttccggaggtttttgaaag gg 2082 3 549 PRT Magnaporthe grisea 3 Met Glu Asn Gly AlaAla Thr Asn Gly Ala Ser Glu Lys Ser His Ser 1 5 10 15 Pro Ser Gln ThrTyr Leu Ser Thr Arg Gly Asp Asp Tyr Gly Leu Ser 20 25 30 Phe Glu Thr ValVal Leu Lys Gly Leu Ala Ala Asp Gly Gly Leu Phe 35 40 45 Leu Pro Glu GluVal Pro Ala Ala Thr Glu Trp Gln Ser Trp Lys Asp 50 55 60 Leu Pro Tyr ThrGlu Leu Ala Val Lys Val Leu Ser Leu Tyr Ile Ser 65 70 75 80 Pro Ala GluVal Pro Thr Glu Asp Leu Arg Ala Leu Val Glu Arg Ser 85 90 95 Tyr Ser ThrPhe Arg Ser Lys Glu Val Val Pro Leu Val Lys Leu Glu 100 105 110 Asp AsnLeu His Leu Leu Glu Leu Phe His Gly Pro Ser Tyr Ser Phe 115 120 125 LysAsp Cys Ala Leu Gln Phe Leu Gly Asn Leu Phe Glu Tyr Phe Leu 130 135 140Thr Arg Lys Asn Lys Gly Lys Glu Gly Lys Asp Arg His His Leu Thr 145 150155 160 Val Val Gly Ala Thr Ser Gly Asp Thr Gly Ser Ala Ala Ile Tyr Gly165 170 175 Leu Arg Asn Lys Lys Asp Val Ser Val Phe Ile Leu His Pro LysGly 180 185 190 Arg Val Ser Pro Ile Gln Glu Ala Gln Met Thr Thr Val LeuAsp Gln 195 200 205 Asn Val His Asn Leu Ala Val Thr Gly Thr Phe Asp AspCys Gln Asp 210 215 220 Ile Val Lys Ala Met Phe Asn Asp Pro Asp Ser AsnAla Thr Leu Lys 225 230 235 240 Leu Gly Ala Val Asn Ser Ile Asn Trp SerArg Ile Leu Ala Gln Ile 245 250 255 Val Tyr Tyr Phe His Ser Tyr Phe SerLeu Ala Arg Ala Ser Pro Glu 260 265 270 Thr Phe Lys Val Gly Asp Lys ValArg Phe Val Thr Pro Thr Gly Asn 275 280 285 Phe Gly Asn Ile Leu Ala GlyTyr Phe Ala Gln Lys Met Gly Leu Pro 290 295 300 Val Asp Lys Leu Val ValAla Thr Asn Glu Asn Asp Ile Leu Asp Arg 305 310 315 320 Phe Trp Lys ThrGly Arg Tyr Glu Lys Lys Pro Ala Ser Pro Glu Glu 325 330 335 Ala Ala GlyGly Leu Pro Gln Asp Gly Val Lys Ala His Glu Glu Gly 340 345 350 Cys LysGlu Thr Leu Ser Pro Ala Met Asp Ile Leu Val Ser Ser Asn 355 360 365 PheGlu Arg Thr Leu Trp Phe Leu Ala Lys Glu Phe Ala Ala Thr Ala 370 375 380Gly Leu Asn Asp Glu Phe Asn Lys Lys Gln Ala Gly Gln Glu Val Val 385 390395 400 Ala Trp Tyr Lys Ser Leu Lys Ala Thr Gly Gly Phe Gly Pro Val His405 410 415 Pro Glu Ile Met Asp Asn Gly Arg Gln Val Phe Glu Ser Glu ArgVal 420 425 430 Ser Asp Thr Gln Thr Leu Glu Met Ile Ala Glu Met Tyr LysAla Thr 435 440 445 Lys Tyr Val Leu Asp Pro His Ser Ala Val Gly Val AlaGly Ala Lys 450 455 460 Arg Ser Met Ser Arg Ala Ser Asn Val Pro His IleAla Leu Ser Thr 465 470 475 480 Ala His Pro Ala Lys Phe Ser Gly Ala ValGlu Leu Ala Leu Lys Asp 485 490 495 Gln Lys Glu Phe Asp Phe Thr Lys GlnVal Leu Pro Glu Asp Phe Val 500 505 510 Gly Leu Ala Glu Lys Glu Lys ArgVal Thr Glu Val Ala Ala Asn Trp 515 520 525 Gln Glu Val Arg Glu Ile ValLys Lys Gln Val Glu Glu Asp Leu Lys 530 535 540 Ala Glu Ser Ser Ala 545

What is claimed is:
 1. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting a fungalThreonine synthase polypeptide with said test compound; and b) detectingthe presence or absence of binding between said test compound and saidfungal Threonine synthase polypeptide; wherein binding indicates thatsaid test compound is a candidate for an antibiotic.
 2. The method ofclaim 1, wherein said Threonine synthase polypeptide is a MagnaportheThreonine synthase polypeptide.
 3. The method of claim 1, wherein saidThreonine synthase polypeptide is SEQ ID NO:
 3. 4. A method fordetermining whether a compound identified as an antibiotic candidate bythe method of claim 1 has antifungal activity, further comprising:contacting a fungus or fungal cells with said antibiotic candidate anddetecting the decrease in growth, viability, or pathogenicity of saidfungus or fungal cells.
 5. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) contacting said testcompound with at least one polypeptide selected from the groupconsisting of: a polypeptide having at least ten consecutive amino acidsof a fungal Threonine synthase, a polypeptide having at least 50%sequence identity with a fungal Threonine synthase, and a polypeptidehaving at least 10% of the activity thereof; and b) detecting thepresence and/or absence of binding between said test compound and saidpolypeptide; wherein binding indicates that said test compound is acandidate for an antibiotic.
 6. A method for determining whether acompound identified as an antibiotic candidate by the method of claim 5has antifungal activity, further comprising: contacting a fungus orfungal cells with said antibiotic candidate and detecting a decrease ingrowth, viability, or pathogenicity of said fungus or fungal cells.
 7. Amethod for identifying a test compound as a candidate for an antibiotic,comprising: a) contacting O-phospho-L-homoserine and water with aThreonine synthase; b) contacting O-phospho-L-homoserine and water withThreonine synthase and said test compound; and c) determining the changein concentration for at least one of the following:O-phospho-L-homoserine, L-threonine, orthophosphate, and water; whereina change in concentration for any of the above substances between steps(a) and (b) indicates that said test compound is a candidate for anantibiotic.
 8. The method of claim 7, wherein said Threonine synthase isa fungal Threonine synthase.
 9. The method of claim 7, wherein saidThreonine synthase is a Magnaporthe Threonine synthase.
 10. The methodof claim 7, wherein said Threonine synthase is SEQ ID NO:
 3. 11. Amethod for determining whether a compound identified as anantibioticcandidate by the method of claim 7 has antifungal activity,further comprising: contacting a fungus or fungal cells with saidantibiotic candidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 12. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting L-threonine and orthophosphate with aThreonine synthase; b) contacting L-threonine and orthophosphate with aThreonine synthase and said test compound; and c) determining the changein concentration for at least one of the following:O-phospho-L-homoserine, L-threonine, orthophosphate, and water; whereina change in concentration for any of the above substances between steps(a) and (b) indicates that said test compound is a candidate for anantibiotic.
 13. The method of claim 12, wherein said Threonine synthaseis a fungal Threonine synthase.
 14. The method of claim 12, wherein saidThreonine synthase is a Magnaporthe Threonine synthase.
 15. The methodof claim 12, wherein said Threonine synthase is SEQ ID NO:
 3. 16. Amethod for determining whether a compound identified as an antibioticcandidate by the method of claim 12 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 17. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) contacting O-phospho-L-homoserine and water with apolypeptide selected from the group consisting of: a polypeptide havingat least 50% sequence identity with Threonine synthase, a polypeptidehaving at least 50% sequence identity with a Threonine synthase andhaving at least 10% of the activity thereof, and a polypeptidecomprising at least 100 consecutive amino acids of a Threonine synthaseb) contacting O-phospho-L-homoserine and water with said polypeptide andsaid test compound; and c) determining the change in concentration forat least one of the following: O-phospho-L-homoserine, L-threonine,orthophosphate, and water; wherein a change in concentration for any ofthe above substances between steps (a) and (b) indicates that said testcompound is a candidate for an antibiotic.
 18. A method for identifyinga test compound as a candidate for an antibiotic, comprising: a)contacting L-threonine and orthophosphate with a polypeptide selectedfrom the group consisting of: a polypeptide having at least 50% sequenceidentity with a Threonine synthase, a polypeptide having at least 50%sequence identity with a Threonine synthase and at least 10% of theactivity thereof, and a polypeptide comprising at least 100 consecutiveamino acids of a Threonine synthase b) contacting L-threonine andorthophosphate, with said polypeptide and said test compound; and c)determining the change in concentration for at least one of thefollowing: O-phospho-L-homoserine, L-threonine, orthophosphate, andwater; wherein a change in concentration for any of the above substancesbetween steps (a) and (b) indicates that said test compound is acandidate for an antibiotic.
 19. A method for identifying a testcompound as a candidate for an antibiotic, comprising: a) measuring theexpression of a Threonine synthase in a fungal cell, cells, tissue, ororganism in the absence of said compound; b) contacting said fungalcell, cells, tissue, or organism with said test compound and measuringthe expression of said Threonine synthase in said fungus or fungal cell;c) comparing the expression of Threonine synthase in steps (a) and (b);wherein a lower expression in the presence of said test compoundindicates that said compound is a candidate for an antibiotic.
 20. Themethod of claim 19 wherein said cell, cells, tissue, or organism is, oris derived from a Magnaporthe fungus or fungal cell.
 21. The method ofclaim 19, wherein said Threonine synthase is SEQ ID NO:
 3. 22. Themethod of claim 19, wherein the expression of Threonine synthase ismeasured by detecting THR4 mRNA.
 23. The method of claim 19, wherein theexpression of Threonine synthase is measured by detecting Threoninesynthase polypeptide.
 24. A method for identifying a test compound as acandidate for an antibiotic, comprising: a) providing cells having oneform of a Threonine synthase gene, and providing comparison cells havinga different form of a Threonine synthase gene, b) contacting said cellsand said comparison cells with a test compound and determining thegrowth of said cells and comparison cells in the presence of the testcompound; wherein a difference in growth between said cells and saidcomparison cells in the presence of said compound indicates that saidcompound is a candidate for an antibiotic.
 25. The method of claim 24wherein the cells are fungal cells.
 26. The method of claim 24 whereinthe cells are Magnaporthe cells.
 27. The method of claim 24 wherein saidform and said comparison form of the Threonine synthase are fungalThreonine synthases.
 28. The method of claim 24, wherein at least oneform is a Magnaporthe Threonine synthase.
 29. The method of claim 24wherein said form and said comparison form of the Threonine synthase arenon-fungal Threonine synthases.
 30. The method of claim 24 wherein oneform of the Threonine synthase is a fungal Threonine synthase, and theother form is a non-fungal Threonine synthase.
 31. A method foridentifying a test compound as a candidate for an antibiotic,comprising: a) providing cells having one form of a gene in theL-threonine biochemical and/or genetic pathway and providing comparisoncells having a different form of said gene. b) contacting said cells andcomparison cells with a said test compound, c) determining the growth ofsaid cells and comparison cells in the presence of said test compound;wherein a difference in growth between said cells and said comparisoncells in the presence of said compound indicates that said compound is acandidate for an antibiotic.
 32. The method of claim 31 wherein thecells are fungal cells.
 33. The method of claim 31 wherein the cells areMagnaporthe cells.
 34. The method of claim 31 wherein said form and saidcomparison form of the L-threonine biosynthesis gene are fungalL-threonine biosynthesis genes.
 35. The method of claim 31, wherein atleast one form is a Magnaporthe L-threonine biosynthesis gene.
 36. Themethod of claim 31 wherein said form and said comparison form of theL-threonine biosynthesis genes are non-fungal L-threonine biosynthesisgenes.
 37. The method of claim 31 wherein one form of the L-threoninebiosynthesis gene is a fungal L-threonine biosynthesis gene, and theother form is a non-fungal L-threonine biosynthesis gene.
 38. A methodfor determining whether a test compound identified as an antibioticcandidate by the method of claim 31 has antifungal activity, furthercomprising: contacting a fungus or fungal cells with said antibioticcandidate and detecting a decrease in growth, viability, orpathogenicity of said fungus or fungal cells.
 39. A method foridentifying a test compound as a candidate for an antibiotic,comprising: (a) providing paired growth media; comprising a first mediumand a second medium, wherein said second medium contains a higher levelof L-threonine than said first medium; (b) contacting an organism withsaid test compound; (c) inoculating said first and second media withsaid organism; and (d) determining the growth of said organism; whereina difference in growth of the organism between said first and secondmedia indicates that said test compound is a candidate for anantibiotic.
 40. The method of claim 39, wherein said organism is afungus.
 41. The method of claim 39, wherein said organism is Magnaporthe.
 42. An isolated polynucleotide comprising a nucleotide sequence thatencodes a polypeptide of SEQ ID NO:
 3. 43. The polynucleotide of claim42 comprising the nucleotide sequence of SEQ ID NO:
 1. 44. An expressioncassette comprising the polynucleotide of claim
 43. 45. The isolatedpolynucleotide of claim 42 comprising a nucleotide sequence of at least50 to at least 95% sequence identity to SEQ ID NO:
 1. 46. An isolatedpolypeptide consisting essentially of the amino acid sequence of SEQ IDNO:
 3. 47. An isolated polypeptide comprising the amino acid sequence ofSEQ ID NO: 3.